1. Shawn T. Leonard MT(ASCP) Blood Centers of the Pacific Antibody Exclusion “Crossing-Out” Interpreting Panel Results

2. Performing selected tests Determining the strength & pattern of agglutination Interpreting results Selecting suitable blood for the patient Antibody Identification Involves YOU as the Expert!

3. Define Antibody Exclusion List advantages of crossing out in Ab Identification Describe limitations of the cross-out process Identify helpful patient and reagent information Discuss AABB Standards on assigning Ab specificity Recognize homozygous vs. heterozygous expression Apply recommended rules for crossing out Objectives

4. Systematic process by which panel results are interpreted to eliminate antibody specificities on the basis of non- reactivity of patient plasma with red cell samples that express the antigen. -AABB Technical Manual 16 th edition “Antibody Exclusion” – Immunohematology definition

5. Commonly known as… “Antibody Exclusion” – Immunohematology definition Crossing-Out

6. 1. Allows accurate ID of antibodies 2. Eliminates additional antibody specificities 3. Provides higher predictive value 4. Supports initial hypothesis 5. Determines additional testing 6. Decreases delay in patient care Advantages of Crossing-Out

7. A first approach May not lead directly to an answer A provisional step Converging evidence ??? Crossing Out

8. Patient’s phenotype Ethnicity of the patient Ethnicity of panel cell donor Age of the panel cell Helpful Information

9. Cross-Out protocol varies by institution Recommended rules for Crossing-Out

10. To increase the probability of correct identification a p-value of <0.05 is observed which gives a 95% confidence interval. What about Ruling-In?

11. Standards for Immunohematology Reference Laboratories-6 th Edition “Assign specificity after demonstrating reactivity with at least two antigen-positive reagent red cell samples and non-reactivity with at least two antigen-negative reagent red cell samples.” AABB IRL Standards

12. Recommended rules for Crossing-Out

13. It is best to rule out an antibody specificity on a cell with a double-dose expression Homozygous alleles- Jk a /Jk a (Double-Dose) express Jk(a+b-) Heterozygous alleles - Jk a /Jk b (Single-Dose) express Jk(a+b+) Rule #1

14. It is best to rule out an antibody specificity on a cell with a double-dose expression Exceptions… K1 and P 1 C or E with Anti-D Rule #1

15. It is better to rule out specificities with two unique cells rather than one By chance, one may not react as well as expected due to: Variant antigen structure Lack of antigen recognition by antibody Weakened antigen expression Antigen degradation Tech error Rule #2

16. “ Reverse of Rule-out ”- for the antibody that shows extreme variability or reacts in more than one phase of testing Anti-P 1 Anti-Le a Anti-Le b Rule #3

17. Antibody Exclusion alone does not define which antibodies are or are not present Additional Techniques : Selected Cells Chemical Treatment Proteolytic Enzymes - Papain, Ficin, Bromelin Sulfhydryl Reagents - DTT & AET Neutralization P 1 & Lewis Rule #4

18. If antibodies to common blood group antigens cannot be ruled out, it may be necessary to consult a Blood Bank Reference Laboratory or provide antigen negative units. When All Else Fails

19. On the following example panels, try crossing out each blood group antigen with at least one homozygous representation. Example Panels

20. CELL RHKellDuffyKiddLewisPMNS CELLGEL IAT IAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1++00+0++++00+0++0+14+ 2++00+++0+0++0+++++24+ 3+0++00+0++00+0++++30 4+00++0+00+000+0+0+40 50+0++0+0++0+0+s+s 0+0+54+ 600+++0++00+0++++++60 7000++++0+++0++++++70 8000++0+0+++0+++0+080 9000++0++0+0+0+++0+90 10000++0+++0+0++0+0+ 0 11++00+0+0++000++++0 4+ AC 0 (Panel A) Example #1

21. CELL RHKellDuffyKiddLewisPMNS CELLGEL IAT IAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1++00+0++++00+0++0+10 2++00+++0+0++0+++++23+ 3+0++00+0++00+0++++33+ 4+00++0+00+000+0+0+40 50+0++0+0++0+0+S+S 0+0+50 600+++0++00+0++++++63+ 7000++++0+++0++++++73+ 8000++0+0+++0+++0+083+ 9000++0++0+0+0+++0+90 10000++0+++0+0++0+0+ 0 11++00+0+0++000++++0 3+ AC 0 (Panel A) Example #2

22. CELL RHKellDuffyKiddLewisPMNS CELLGEL IAT IAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 12000++0+0++00+++++013+ 13000++++++0+0++S+S +00+20 14000++0++00+0++++0+30 15+0++00++0+++0+S+S ++0+42+ 16+0++00+++0+0+0++++53+ 17+0++00+0++0+0++0++63+ 18++00+0+0++0+000+0+70 19++00+0++00+0+++0+083+ 20+++0+0++0++0++S+S ++0+92+ 210+0++0+0+++0+++00+100 22++00++00++00++++++113+ AC 0 (Panel B) Example #2

23. CELL RHKellDuffyKiddLewisPMNS CELLGEL IAT IAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1++00+0++++00+0++0+14+ 2++00+++0+0++0+++++24+ 3+0++00+0++00+0++++34+ 4+00++0+00+000+0+0+44+ 50+0++0+0++0+0+s+s 0+0+50 600+++0++00+0++++++60 7000++++0+++0++++++70 8000++0+0+++0+++0+080 9000++0++0+0+0+++0+90 10000++0+++0+0++0+0+ 0 11++00+0+0++000++++0 4+ AC 0 (Panel A) Example #3

24. CELL RHKellDuffyKiddLewisPMNS CELLLISS 37 o LISSIAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1+++0+0++0+00+++++0100 2++00+0+0+0+0+++00+21+1+ 3+0++00+0+0+0+00++0300 4+00++0+00++0+++++0400 50+0++0++00+0+++0+051+1+ 600+++0+0+++0++++0+600 7000++++0+0+0+++0++71+1+ 8000++0++0+0+000+++800 9000++0++0+++00+00+91+1+ 10++00+++0++00+++++0 00 11000++0+0++00+00++0 00 AC 00 Example #4

25. CELL RHKellDuffyKiddLewisPMNS CELLLISSIAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1+++0+0++++0+0+++0+1w+ 2++00+++0+++0000+0+20 3+0++00+0++000+0+++30 4+00++0++0++0++0+0041+ 50+0++0+++0+0++++0+5w+ 600+++0++00+0++++0+60 7000++++0+0++00+0+070 8000++0++00+00++0+08w+ 9000++0+00++0++S+S 0+0+92+ 10++00++0++0+0+++0++ 0 11++00+0++0+++00++0+ 0 AC 0 Example #5

26. CELL RHKellDuffyKiddLewisPMNS CELLRT15’ P 1 Neut.RT15’ DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1+++0+0++++0+0+++0+11+0 2++00+++0+++0000+0+200 3+0++00+0++000+0+++31+0 4+00++0++0++0++0+0041+0 50+0++0+++0+0++++0+51+0 600+++0++00+0++++0+61+0 7000++++0+0++00+0+0700 8000++0++00+00++0+081+0 9000++0+00++0++S+S 0+0+92+0 10++00++0++0+0+++0++ 1+0 11++00+0++0+++00++0+ 00 AC (RT & P 1 Neut.) Example #5

27. CELL RHKellDuffyKiddLewisPMNS CELLLISS 37 o LISSIAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1+++0+0+0+0+0+++0++11+2+ 2++00+0+0++00+00+0+202+ 3+0++00+0+0+0++0+0+31+2+ 4+00++0++0++0++++0+400 50+0++0++++00+++0++502+ 600+++0++00+0++++0+61+2+ 7000++++0++++0+++++700 8000++0++0+++000+++800 9000++0++0+0+0+++++902+ 10++00++++++00++0+0+ 02+ 11++00+0++00+0+0+0+0 00 AC 00 Example #6

28. CELL RHKellDuffyKiddLewisPMNS CELLLISSIAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1++00++++++0+0+0++013+ 2++00+0+0+0+0++0+++22+ 3+0++00+000+0+0+0++32+ 4+00++0+00+00++++0040 50+0+++++0+00+00++052+ 600+++0+0+++0++0+++64+ 7000+++++0+++0+++0+72+ 8000++0++00+0+++0++80 9000++0+0+++0+++0+092+ 10++++00+0+++00+0+0+ 4+ 11++00+0+00+0+0+0+0+ 0 AC 0 Example #7

29. CELL RHKellDuffyKiddLewisPMNS LISSIATENZ IAT IATDTTIAT DCEceKk Fy a Fy b Jk a Jk b Le a Le b P1P1P1P1MNSs 1++00++++++0+0+0++03+2+2+ 2++00+0+0+0+0++0+++2+02+ 3+0++00+000+0+0+0++2+3+2+ 4+00++0+00+00++++00000 50+0+++++0+00+00++02+2+0 600+++0+0+++0++0+++4+3+4+ 7000+++++0+++0+++0+2+2+0 8000++0++00+0+++0++000 9000++0+0+++0+++0+02+02+ 10++++00+0+++00+0+0+4+3+4+ 11++00+0+00+0+0+0+0+000 AC0 (Chemicals) Example #7